Over the past few years it has been observed that several types of molecules can bind to DNA and disrupt the function of DNA. Some of these molecules are presently used as antimicrobials and others are used as cancer chemotherapeutics. In the former case, the microbe is inhibited from replicating, and in the latter case, the cancer cell is inhibited from replicating.
DNA-binding molecules may be categorized based on the type of interaction that occurs between the DNA and the DNA-binding molecule. These types of interactions include electrostatic interactions, covalent binding, intercalation, and reversible groove-binding (most commonly in the minor groove of the B-form DNA helix). Certain DNA-binding molecules involve more than one of these interactions, such as diamidino-2-phenylindole (DAPI), which is able to both intercalate and bind within the grooves of DNA.
Of the four categories of DNA-binding molecules the intercalators and the groove binders are the most common. Intercalators function by causing a local fixation withouut unwinding and extension of the DNA helix, with the intercalator positioning itself between the base pairs. Common antibiotics which are intercalators are anthracyclines, nogalamycin, and actinomycin. The groove binders function by binding in the grooves of the helix. The most common groove binders are the N-methylpyrrole peptides, such as netropsin and distamycin.
The distortion caused by groove binding is significantly less than that associated with intercalators. As a result, intercalating agents have attracted more attention than groove binders as therapeutic agents.
The stabilization of short duplexes by intercalating agents has been recognized for many years. This phenomenon of stabilization has been applied to the antisense field. A duplex formed by an antisense molecule and its DNA or RNA target is significantly stabilized by free intercalating agents, thereby enhancing the effectiveness of the antisense molecule. Such intercalating agents have been found to stabilize such duplexes even when covalently bound to the antisense molecule.
As with intercalation agents, it has been observed that free distamycin and netropsin when added to a DNA duplex will stabilize that duplex. (They will not, however, stabilize a DNA-RNA duplex.) These molecules are believed to displace the natural hydration from AT-rich regions of the minor groove of the duplex. At this AT-rich region of the DNA duplex, the free distamycin or netropsin form bifurcated hydrogen bonds with adenine N-3 and thymine O-2 atoms and numerous van der Waals contacts with various atoms in the nucleotide backbone. These atomic interactions stabilize the DNA-distamycin or netropsin structure and, in turn, effectively strengthen the interaction of the two DNA strands.
In order to interfere with the replication or transcription of a specific DNA molecule researchers have used a combination of free netropsin or distamycin and antisense oligonucleotides having AT rich regions. The antisense oligonucleotide binds to the specific DNA sequence and the free netropsin or distamycin interacts with the minor groove and strengthens the DNA antisense oligonucleotide complex, thereby making the antisense molecule more effective at inhibiting the formation of a transcription bubble and inhibiting transcription.
It recently was attempted to covalently attach N-methylpyrrolecarboxamides (MPCs) to short oligodeoxynucleotides to determine whether the tethered MPCs could still function to stabilize a DNA-DNA duplex. Sinyakov, A. N., et al., J Am. Chem. Soc., 117:4995-4996, (1995). The short oligodeoxynucleotides used were either poly A or poly T, and the target DNA likewise was poly A or poly T sequences. Neither netropsin nor distamycin were attached to these oligodeoxynucleotides. Instead, synthetic MPCs were used, having 2, 3, 4, or 5 methylpyrrolecarboxamide moieties. The MPC was attached to the oligodeoxynucleotide by the carbon atom at the 3C position of the pyrrole moiety of the N-terminal N-methylpyrrolecarboxamide. The following structure is an example of the complexes disclosed in Sinyakov et al.: ##STR1## wherein n=2-5 and wherein X is a linker.
The MPC-oligodeoxynucleotide complex was hybridized with DNA and the resultant MPC-oligodeoxynucleotide/DNA duplex was subjected to melting conditions to determine the melting temperature of the complex. The melting temperature was compared to the melting temperature of the DNA duplex in the absence of N-methylpyrrolecarboxamide and to the melting temperature of the DNA duplex in the presence of a free N-methylpyrrolecarboxamide, in particular, free distamycin.
A number of observations emerged from the results of this study. Firstly, the tethered MPCs stabilized the duplexes as in all instances versus the duplex in the absence of any free distamycin. Secondly, the degree of stabilization was a function of MPC peptide length, with an MPC having two N-methylpyrrolecarboxamide moieties showing the least stabilization and an MPC having 5 N-methylpyrrolecarboxamide moieties showing the most stabilization. The difference between MPCs with 2 and 3 N-methylpyrrolecarboxamide moieties was most pronounced with 3 such moieties increasing the melting temperature by about nine degrees more than the melting temperature with two such moieties. Thirdly, when the MPCs were tethered to poly A deoxynucleotides, only the covalent complex using an MPC having 5 N-methylpyrrolecarboxamide moieties had a better stabilization effect than free distamycin. When the MPCs were tethered to poly T deoxynucleotides, only the covalent complexes using an MPC having 4 and 5 N-methylpyrrolecarboxamide moieties had a substantially better stabilization effect versus free distamycin (.DELTA.T.sub.m =13 and 18 respectively), whereas the MPC with 2 such moieties did not work as well as free distamycin and the MPC with 3 such moieties worked about the same as free distamycin (.DELTA.T.sub.m .apprxeq.5). Finally, the MPC peptide was found to be more effective at stabilizing the complex when it was covalently attached to poly(dT).sub.8 than to poly(dA).sub.8.